Thermal stability of bovine-brain myelin membrane

The thermal behaviour of bovine-brain myelin membrane has been studied by high-sensitivity differential scanning calorimetry, Fourier-transform infrared spectroscopy and thermal gel analysis. Spectroscopic results indicate that protein transitions take place between 60°C and 90°C, while thermal gel analysis has provided the thermal denaturation profiles of myelin proteolipid, DM-20 protein and the Wolfgram Fraction. An irreversible calorimetric transition centred at 80.3 ± 0.2°C with a specific enthalpy of 4.7 ± 0.6 J/g of total protein has been assigned to the thermal denaturation of myelin proteolipid and DM-20 protein. The effects of the myelin storage conditions, scan rate, ionic strength and pH on this calorimetric transition have also been investigated. The thermal transition of the proteolipid practically disappears after treatment of the myelin with different amounts of chloroform-methanol 2:1 (v/v), a treatment which is generally used in proteolipid purification. On the other hand, the addition of several detergents to myelin only causes minor modifications to this transition, which then occurs at about 70°C, with a specific enthalpy of between 2.5 and 3.6 J/g of total protein. These results appear to show that detergents preserve the native conformation of the proteolipid far more than do organic solvents. Hence the use of detergents would seem to be the appropriate method for proteolipid purification.Abbreviations DSC Differential scanning calorimetry - TGA Thermal gel analysis - FTIR Fourier-transform infrared spectroscopy - PLP Proteolipid protein - MBP Myelin basic protein - DM-20 Protein DM-20 - WF Wolfgram fraction - BSA Bovine serum albumine - SDS Sodium dodecyl sulfate - ANSA 4-amino-3-hydroxynaphthalene-1-sulphonic acid - OG -d-glucopyranoside - PAGE Polyacrylamide gel electrophoresis - Chaps 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate - CNS Central nervous system Correspondence to: P. L. Mateo     

pH and ligand binding modulate the strength of protein-protein interactions in the Ca(2+)-ATPase from sarcoplasmic reticulum membranes.

The Ca(2+)-ATPase from sarcoplasmic reticulum (SR) membranes couples the Ca(2+) transport to ATP hydrolysis through phosphorylation in its cytoplasmic catalytic domain. Interactions between protein domains and the role of monomer-monomer interactions remain unclear. Here, we report a differential scanning calorimetric study of the thermal unfolding of this protein. In the pH range 6-8, thermal unfolding of the Ca(2+)-ATPase in glycogen phosphorylase-free SR membranes shows a major endothermic peak with a critical temperature midpoint ranging between 51 and 55 degrees C, depending on pH, Ca(2+), Mg(2+)-ADP and KCl concentrations. The enthalpy change of the overall unfolding process ranged between 250 and 300 kcal/mol of Ca(2+)-ATPase monomer. Thermal denaturation of the Ca(2+)-ATPase in SR membranes is well fitted to an irreversible process that can be rationalized in terms of a non-two state process, N (native)right harpoon over left harpoon I (intermediate)-->D (denatured). Thermodynamic analysis show that this protein has a compact structure, implying a tight structural interconnection between catalytic and Ca(2+) transport domains. The apparent cooperative unit, defined by the van 't Hoff enthalpy to the overall unfolding enthalpy ratio, increased from 1.1 at pH 6 to 1.8 at pH 8, showing that monomer-monomer interactions are stronger at weakly basic pH than at weakly acidic pH. While micromolar Ca(2+) concentrations had only a weak effect on the cooperativity of the unfolding process, this is clearly increased by millimolar Mg(2+)-ADP. In addition, high ionic strength lowered the apparent cooperative unit to approximately 1.0 in the pH range 6-8. Taken together, these results suggest that protein-protein interactions are altered by variables that modulate the catalytic activity of this enzyme.     

Purification and characterization of glycogen phosphorylase A and B from the freeze-avoiding gall moth larvae Epiblema scudderiana

The active a and inactive b forms of glycogen phosphorylase from cold-hardy larvae of the gall moth, Epiblema scudderiana, were purified using DEAE⁺ ion exchange and 3-5-AMP-agarose affinity chromatography. Maximum activities for glycogen phosphorylases a and b were 6.3±0.74 and 2.7±0.87 mol glucose-1-P·min^-1·g wet weight^-1, respectively, in -4°C-acclimated larvae. Final specific activities of the purified enzymes were 396 and 82 units·mg protein^-1, respectively. Both enzymes were dimers with native molecular weights of 215000±18000 for glycogen phosphorylase a and 209000±15000 for glycogen phosphorylase b; the subunit molecular weight of both forms was 87000±2000. Both enzymes showed pH optima of 7.5 at 22°C and a break in the Arrhenius relationship with a two- to four-fold increase in activation energy below 10°C. Michaelis constant values for glycogen at 22°C were 0.12±0.004 mg·ml^-1 for glycogen phosphorylase a and 0.87±0.034 mg·ml^-1 for glycogen phosphorylase b; the Michaelis constant for inorganic phosphate was 6.5±0.07 mmol·l^-1 for glycogen phosphorylase a and 23.6 mmol·l^-1 for glycogen phosphorylase b. Glycogen phosphorylase b was activated by adenosine monophosphate with a K _a of 0.176±0.004 mmol·l^-1. Michaelis constant and K _a values decreased by two- to fivefold at 5°C compared with 22°C. Glycerol had a positive effect on the Michaelis constant for glycogen for glycogen phosphorylase a at intermediate concentrations (0.5 mol·l^-1) but was inhibitory to both enzyme forms at high concentrations (2 mol·l^-1). Glycerol production as a cryoprotectant in E. scudderiana larvae is facilitated by the low temperature-simulated glycogen phosphorylase b to glycogen phosphorylase a conversion and by positive effects of low temperature on the kinetic properties of glycogen phosphorylase a. Enzyme shut-down when polyol synthesis is complete appears to be aided by strong inhibitory effects of glycerol and KCl on glycogen phosphorylase b.Abbreviations E _a activation energy - GPa glycogen phosphorylase a - GPb glycogen phosphorylase b - h Hill coefficient - I ₅₀ concentration of inhibitor that reduces enzymes velocity by 50% - K _a concentration of activator that produces half-maximal activation of enzyme activity - K _m Michaelis-Menten substrate affinity constant - MW molecular weight - PEG polyethylene glycol - P_i morganic phosphate - SDS PAGE sodium dodecyl sulphate polyacrylamide gel electrophoresis - V _max enzyme maximal velocity     

Phosphorylation-induced conformational changes in the phosphorylaseab hybrid as revealed by resolution of pyridoxal 5′-phosphate with imidazole citrate and cysteine

Summary The accessibility of pyridoxal 5′-phosphates of the phosphorylaseab hybrid to resolution by imidazole citrate and cysteine was studied and compared with that of theb anda forms. Promotion of resolution of phosphorylated forms by raising the temperature or in the presence of glycogen indicates that the resistance of phosphorylasea andab to resolution at 0°C is due rather to their tetrameric state than their phosphorylation-related active conformation. The pattern of resolution of theab hybrid was similar to that of thea and differed from that of theb forms in that it occurred at 30°C and 37°C but not at 0°C, moreover, it did not show first-order kinetics. On the other hand, inhibition of resolution by ligands binding to the nucleotide site of phosphorylase reflected an intermediate sensitivity of theab form between that of theb anda forms. We conclude that partial phosphorylation of phosphorylaseb elicits conformational change(s) in both subunits which influence the monomer-monomer interactions and resolution of pyridoxal 5′-phosphates. Resistance ofab hybrid to monomerizing agents as imidazole citrate, comparable to that of other forms, argues for its stability, ruling out its reshuffling into mixtures of phosphorylaseb anda.     

Circular dichroism spectra of human hemoglobin reveal a reversible structural transition at body temperature

Previously we have shown that human red blood cells (RBCs) undergo a sudden change from blocking to passing through a 1.3±0.2-µm micropipette when applying an aspiration pressure of 2.3 kPa at a critical transition temperature (T_c=36.4±0.3 °C). Low-shear viscosity measurements suggested that changes in the molecular properties of hemoglobin might be responsible for this effect. To evaluate structural changes in hemoglobin at the critical temperature, we have used circular dichroism (CD) spectroscopy. The thermal denaturation curves of human hemoglobin A (HbA) and hemoglobin S (HbS) upon heating between 25 and 60 °C were non-linear and showed accelerated denaturation between 35 and 39 °C with a midpoint at 37.2±0.6 °C. The transition was reversible below 39 °C and independent of solution pH (pH 6.8–7.8). It was also independent of the oxygenation state of hemoglobin, since a sample that was extensively deoxygenated with N₂ showed a similar transition by CD. These findings suggest that a structural change in hemoglobin may enable the cellular passage phenomenon as well as the temperature-dependent decrease in viscosity of RBC solutions.     

A primer independent activity of rabbit muscle phosphorylaseb

Summary Rabbit muscle phosphorylaseb was found to be capable of forming protein bound±-1,4 glucosyl chains upon incubation of the enzyme with appropriate concentrations of glucose-1-phosphate with no primer addition (unprimed synthesis). This activity would only be present in a small fraction of the total muscle phosphorylaseb activity, as judged from the high concentrations of enzyme which are required to demonstrate the occurrence of unprimed synthesis. Polyacrylamide gel electrophoresis shows the presence of a phosphorylase isoenzyme capable of accepting glucosyl moieties, giving rise to a glucosylated protein enzymatically active in the chain lengthening of its own glucan.Dedicated to ProfessorLuis F. Leloir on the occasion of his 70^th birthday.     

Regulation of glycogen phosphorylase in fat body ofCecropia silkmoth pupae

Summary Glycogen phosphorylase of pupal fat body of the silkmoth,Hyalophora cecropia, and its activation by different stimuli have been studied. Spectrophotometric assay in the direction of glycogenolysis, used in most of the experiments, indicated higher amounts of phosphorylasea than assay by release of P_i from glucose-1-phosphate; both assays, however, estimated changes in proportion of phosphorylasea equally. TheK _ms for P_i were estimated as 5 mM for phosphorylasea in the absence of AMP and 18 mM for phosphorylaseb with 2 mM AMP.When diapausing pupae were held at 4°C, fat body phosphorylase was quickly activated by conversion to thea form up to about 50% of the total, and then declined again after 30 days, when glycerol had accumulated in the hemolymph. Cold activation in vivo was quickly reversed at 25°C. Removal of the brain did not prevent cold activation. After storage at 15°C, sensitivity to cold activation was diminished. Locusts and crickets also showed activation of phosphorylase after chilling.Exposure of fat body to air, transfer to Ringer solution, or physical agitation, caused activation of phosphorylase which is classed as shock activation. After about 1 h incubation in Ringer at 25°C, this effect reversed spontaneously. Activation also occurred in fat body in vitro after transfer to 0°C (cold activation), and was reversed at 25°C. The previously reported inhibition of activation by glycerol, however, could not be consistently reproduced.In fat body homogenates, phosphorylaseb was converted to phosphorylasea by incubation with ATP and Mg²⁺, which indicates activity of phosphorylase kinase. In preparations treated with Sephadex G-25 and then incubated, the reverse conversion took place, which was inhibited by fluoride, and indicates activity of phosphorylase phosphatase.Cyclic AMP added to fat body in vitro, or theophylline either in vivo or in vitro, stimulated the activation of phosphorylase. In fat body in vitro, shock activation was paralleled by elevation of tissue cyclic AMP, whereas cold activation was not. Cyclic GMP did not stimulate activation, and showed no significant changes in tissue levels.It is concluded that the conversion of silkmoth pupal fat body phosphorylaseb to phosphorylasea can be stimulated by a shock-initiated mechanism involving cyclic AMP and a distinct cold-initiated mechanism independent of cyclic AMP.Abbreviations DTT dithiothreitol - cyclic AMP 3,5-cyclic adenosine monophosphate - cyclic GMP 3,5-cyclic guanosine monophosphate - P _i inorganic phosphate This investigation was begun in the Department of Biology, Yale University, New Haven, Connecticut, USA     

Bio-Beads: An Efficient Strategy for Two-Dimensional Crystallization of Membrane Proteins

This work establishes the potential of Bio-Beads as a simple alternative to conventional dialysis for removing detergent and for obtaining 2D crystals of integral membrane proteins useful for structure analysis by electron crystallography. Kinetic and equilibrium aspects of removal of different detergents by adsorption onto hydrophobic Bio-Beads SM2 have been systematically investigated and extended to 2D crystallization of different prototypic membrane proteins, including: (a) Ca²⁺ATPase from sarcoplasmic reticulum; (b) melibiose permease fromEscherichia coli;(c) cytochromeb₆ffromChlamydomonas reinhardtii.Different crystals could be produced from all protein preparations, with optical diffraction down to 20–25 Å in negative stain.     

Interaction of the local anesthetics dibucaine and tetracaine with sarcoplasmic reticulum membranes. Differential scanning calorimetry and fluorescence studies

The local anesthetics dibucaine and tetracaine inhibit the (Ca2+ + Mg2+)-ATPase from skeletal muscle sarcoplasmic reticulum [DeBoland, A. R., Jilka, R. L., & Martonosi, A. N. (1975) J. Biol. Chem. 250, 7501-7510; Suko, J., Winkler, F., Scharinger, B., & Hellmann, G. (1976) Biochim. Biophys. Acta 443, 571-586]. We have carried out differential scanning calorimetry and fluorescence measurements to study the interaction of these drugs with sarcoplasmic reticulum membranes and with purified (Ca2+ + Mg2+)-ATPase. The temperature range of denaturation of the (Ca2+ + Mg2+)-ATPase in the sarcoplasmic reticulum membrane, determined from our scanning calorimetry experiments, is ca. 45-55 degrees C and for the purified enzyme ca. 40-50 degrees C. Millimolar concentrations of dibucaine and tetracaine, and ethanol at concentrations higher than 1% v/v, lower a few degrees (degrees C) the denaturation temperature of the (Ca2+ + Mg2+)-ATPase. Other local anesthetics reported to have no effect on the ATPase activity, such as lidocaine and procaine, did not significantly alter the differential scanning calorimetry pattern of these membranes up to a concentration of 10 mM. The order parameter of the sarcoplasmic reticulum membranes, calculated from measurements of the polarization of the fluorescence of diphenylhexatriene, is not significantly altered at the local anesthetic concentrations that shift the denaturation temperature of the (Ca2+ + Mg2+)-ATPase.(ABSTRACT TRUNCATED AT 250 WORDS)     

A robust method to screen detergents for membrane protein stabilization,revisited

This report is a follow up of our previous paper (Lund, Orlowski, de Foresta, Champeil, le Maire and Møller (1989), J Biol Chem 264:4907–4915) showing that solubilization in detergent of a membrane protein may interfere with its long-term stability, and proposing a protocol to reveal the kinetics of such irreversible inactivation. We here clarify the fact that when various detergents are tested for their effects, special attention has of course to be paid to their critical micelle concentration. We also investigate the effects of a few more detergents, some of which have been recently advertised in the literature, and emphasize the role of lipids together with detergents. Among these detergents, lauryl maltose neopentyl glycol (LMNG) exerts a remarkable ability, even higher than that of β-dodecylmaltoside (DDM), to protect our test enzyme, the paradigmatic P-type ATPase SERCA1a from sarcoplasmic reticulum. Performing such experiments for one's favourite protein probably remains useful in pre-screening assays testing various detergents.     

Sarcoplasmic reticulum and excitation-contraction coupling at 20 and 10 °C in rainbow trout myocardium

Summary Isometric force and series membrane potential were recorded in isolated ventricular strips from rainbow trout at 20 and 10 °C. Preparations were electrically stimulated to contract at either 0.5 or 0.2 Hz. Single extrastimulations elicited a twitch force which diminished when the preceding diastole was shortened below the regular value. The stimulation following this extra stimulation evoked no potentiation of force. Apart from a marginal effect on the post extrasystolic force at 20 °C, ryanodine did not affect either of these responses or the steady-state force at 0.5 Hz. At 0.2 Hz the steady-state force was somewhat depressed by ryanodine at 20 but not at 10 °C. In contrast, extrastimulations preceded by diastoles of up to 1 h more than doubled extrasystolic force at 20 °C. This effect was removed by ryanodine. Both the potentiations and the effect of ryanodine were strongly reduced at 10 °C. Apparently, temperature acts on the release of Ca²⁺ from the sarcoplasmic reticulum, since Ca²⁺ seems to be taken up at both temperatures. Hence, at both 20 and 10 °C, the contractures evoked in a solution inhibiting sarcolemmal Ca²⁺ transfer and releasing Ca²⁺ from the sarcoplasmic reticulum were diminished by pretreatment with 15 mM caffeine. Action potential duration at 20 °C was less than half of that at 10 °C. At both temperatures it tended to be prolonged by periods of prolonged rest. No effect of ryanodine on action potential configuration was detected. The results suggest that trout myocardial sarcoplasmic reticulum, although powerful at unphysiologically low stimulation rates, does not partake in the beat-to-beat regulation of force at heart rates encountered in vivo.Abbreviations ESF extrasystolic force - SR sarcoplasmic reticulum - v _F maximal rate of force development - v _R maximal rate of relaxation - TPF time to peak force - TR _0.5 time for half relaxation - TTF duration of force development     

Cardiac force-interval relationship,adrenaline and sarcoplasmic reticulum in rainbow trout

The force-interval relationship was examined at 20 and 10 °C in electrically paced atrial and ventricular tissue of rainbow trout,Oncorhynchus mykiss, regarding dependence on the sarcoplasmic reticulum and influence of adrenaline. In both tissues, adrenaline (10^-6 mol·l^-1) doubled control force developed at 0.5 Hz. In atrial but not in ventricular tissue it also shortened the diastolic interval needed for recovery of a given fraction of the control force. In atrial tissue and in ventricular tissue at 20 °C, the fraction of force recovered in the presence of adrenaline was diminished by 10 mol·l^-1 of ryanodine, a specific inhibitor of the sarcoplasmic reticulum. In atrial tissue not exposed to adrenaline and in ventricular tissue at 10 °C irrespective of adrenaline, ryanodine did not affect recovery. In atrial but not in ventricular tissue it also diminished control force. In conclusion, the cardiac sarcoplasmic reticulum of trout seems to support force development during adrenaline dependent increases in heart rate, and in atrial tissue also the force at steady state.Abbreviations E-C coupling excitation-contraction coupling - P-R potential - SR sarcoplasmic reticulum - SE standard error of the mean     

Acetylcholinesterase in membrane fractions derived from sarcotubular system of skeletal muscle: presence of monomeric acetylcholinesterase in sarcoplasmic reticulum and transverse tubule membranes

Microsomes were isolated from white rabbit muscle and separated into several fractions by centrifugation in a discontinuous sucrose density gradient. Four membrane fractions were obtained namely surface membrane, light, intermediate and heavy sarcoplasmic reticulum. The origin of these microsomal vesicles was investigated by studying biochemical markers of sarcoplasmic reticulum and surface and T-tubular membranes. The transverse tubule derived membranes were further purified by using a discontinuous sucrose density gradient after loading contaminating light sarcoplasmic reticulum vesicles with calcium phosphate in the presence of ATP. All membrane preparations displayed acetylcholinesterase activity (AChE, EC 3.1.1.7), this being relatively more concentrated in T-tubule membranes than in those derived from sarcoplasmic reticulum. The membrane-bound AChE of unfractioned microsomes notably increased its activity by aging, treatment with detergents and low trypsin concentrations indicating that the enzyme is probably attached to the membrane in an occluded form, the unconstrained enzyme displaying higher activity than the vesicular acetylcholinesterase.Sedimentation analysis of Triton-solubilized AChE from different membrane fractions revealed enzymic multiple forms of 13.5S, 9–10S and 4.5–4.8S, the lightest form being the predominant one in all membrane preparations. Therefore, in both sarcoplasmic reticulum and T-tubule membrane the major component of AChE appears to be a membrane-bound component, probably a G₁ form.     

An Irreversible and Kinetically Controlled Process: Thermal Induced Denaturation of <Emphasis Type="SmallCaps">L</Emphasis>-2-Hydroxyisocaproate Dehydrogenase from <Emphasis Type="Italic">Lactobacillus confusus</Emphasis>

The thermal denaturation of Lactobacillus confusus l-2-Hydroxyisocaproate Dehydrogenase (l-HicDH) has been studied by Differential Scanning Calorimetry (DSC). The stability of this enzyme has been investigated at different pH conditions. The results of this study indicate that the thermal denaturation of this enzyme is irreversible and the T _m is dependent on the scan-rate, which suggests that the denaturation process of l-HicDH is kinetically determined. The heat capacity function of l-HicDH shows a single peak with the T _m values between 52.14°C and 55.89°C at pH 7.0 at different scan rates. These results indicate that the whole l-HicDH could unfold as a single cooperative unit, and intersubunit interactions of this homotetrameric enzyme must play a significant role in the stabilization of the whole enzyme. The rate constant of the unfolding is analyzed as a first order kinetic constant with the Arrhenius equation, and the activation energy has been calculated. The variation of the activation energy values obtained with different methods does not support the validity of the one-step irreversible model. The denaturation pathway was described by a three-state model, N → U → F, in which the dissociation of the tetramer takes place as an irreversible step before the irreversible unfolding of the monomers. The calorimetric enthalpy associated with the irreversible dissociation and the calorimetric enthalpy associated with the unfolding of the monomer were obtained from the best fitting procedure. Thermal unfolding of l-HicDH was also studied using Circular Dichroism (CD) spectroscopy. Both methods yielded comparable values.     

Denaturation ofBacillus stearothermophilus α-amylase by urea and detergents

Denaturation ofBacillus stearothermophilus -amylase by urea and detergents was investigated for the purpose of understanding the mechanism of denaturation of this enzyme. The enzyme was extremely resistant to denaturation by detergents at 60° C, either in the presence or absence of added calcium. Addition of EDTA was necessary to obtain denaturation by detergents. The rate of denaturation of the -amylase by urea was strongly dependent on the incubation pH and presence or absence of calcium ions. Calcium-binding groups were shown to have pKa values of 5.5 for exogenous calcium and 4.7 to 4.8 for endogenous calcium. A mechanism is proposed for the denaturation ofBacillus stearothermophilus -amylase.     

Structural stability of the erythrocyte anion transporter, band 3, in native membranes and in detergent micelles.

The exothermic thermal denaturation transition of band 3, the anion transporter of the human erythrocyte membranes, has been studied by differential scanning calorimetry, in ghost membranes and in nonionic detergent micelles. In detergent micelles the transmembrane domain of band 3 gave an irreversible denaturation transition (C transition). However, no thermal transition was observed for the N-terminal cytoplasmic domain when band 3 was solubilised in detergent micelles. A reduction in enthalpy (190-300 kcal mol-1) with an accompanying decrease in thermal denaturation temperatures (48-60 degrees C) for the C transition was observed in detergent solubilised band 3 when compared with ghost membranes. Unlike ghost membranes, two thermal transitions for band 3 in detergent micelles were observed for the C transition when in the presence of excess covalent inhibitor, 4,4'-diisothiocyanostilbene-2,2'-disulphonate (DIDS), which derive from the thermal unfolding of a single protein with two different thermal stabilities; DIDS-stabilised (75 degrees C) and DIDS-insensitive (62 degrees C). A reduction in the denaturation temperature for the transmembrane domain of band 3 was observed when compared with intact band 3 although no significant differences was observed in the corresponding enthalpy values. This indicates some cooperativity of the two domains of band 3 in maintaining the transmembrane conformation. The results presented in this study show that detergents of intermediate micelle size (e.g. Triton X-100 and C12E8) are required for optimal thermal stability of band 3.     

Chloride and osmotic contractures in skinned frog muscle fibers

Summary Single contractures were elicited in segments of skinned frog muscle fibers when the segments were moved from relaxing-loading solutions to various test solutions. The effective test solutions produced an increase in the concentration of chloride ions in the myofilament space, [Cl] _ms , and/or presumably caused the sarcoplasmic reticulum to undergo a change in volume. The contractures were quantified in terms of their maximum tension and time-integral. Two outer segments from each fiber underwent a contracture in a control solution (chloride ions were substituted for all of the methanesulfonate ions in the relaxing solution). The mean values of tension and area in the control contractures of each fiber were divided into the corresponding values from a test contracture obtained in the central segment of the same fiber. Test contractures obtained upon increasing [Cl] _ms and increasing the product, [K] _ms ×[Cl] _ms , were compared to contractures that were obtained by increasing [Cl] _ms while keeping [K] _ms ×[Cl] _ms constant. The former contractures were greater in magnitude for a given [Cl] _ms . Whereas the former solutions may have caused an increase in the volume of the sarcoplasmic reticulum and altered the electrical potential across the membranes of the sarcoplasmic reticulum as well, only a change in potential was presumed to have occurred in the latter solutions. Other types of contractures were investigated to show that both swelling of the sarcoplasmic reticulum and changes in the electrical potential of its membranes can cause release of calcium ions and elicit contractures in skinned fibers.     

Kinetics of Denaturation of Rabbit Skeletal Muscle Glycogen Phosphorylase b by Guanidine Hydrochloride

The kinetics of denaturation and aggregation of rabbit muscle glycogen phosphorylase b in the presence of guanidine hydrochloride (GuHCl) have been studied. The curve of inactivation of phosphorylase b in time includes a region of the fast decline in the enzymatic activity,an intermediate plateau,and a part with subsequent decrease in the enzymatic activity. The fact that the shape of the inactivation curves is dependent on the enzyme concentration testifies to the dissociative mechanism of inactivation. The dissociation of phosphorylase b dimers into monomers in the presence of GuHCl is supported by sedimentation data. The rate of phosphorylase b aggregation in the presence of GuHCl rises as the denaturant concentration increases to 1.12 M; at higher concentration of GuHCl, suppression of aggregation occurs. At rather low concentration of the protein (0.25 mg/ml), the terminal phase of aggregation follows the kinetics of a monomolecular reaction (the reaction rate constant is equal to 0.082 min^–1;1 M GuHCl, 25°C). At higher concentration of phosphorylase b (0.75 mg/ml), aggregation proceeds as a trimolecular reaction.     

Daily and seasonal cycles of body temperature and aspects of heterothermy in the hedgehog Erinaceus europaeus

Summary Intra-abdominal temperature-sensitive radio transmitters were used to collect more than 350 sets of body temperature (T _b ) data from 23 captive adult hedgehogs over a 3-year period. Each data set comprised measurements made every 1/2 h for 24-h periods. Between 20 and 60 such data sets were recorded every calendar month, and a total of 17400 measurements of T _b were collected. The hedgehogs were exposed to natural environmental conditions at 57°N in NE Scotland. Hedgehogs showed seasonal changes in mean daily euthermic T _b ,with a July maximum of 35.9±0.2°C, a September minimum of 34.7±0.9°C, and a marked circadian T _b cycle that correlates closely with photoperiod. Maximal T _b occurred within 2 h of midnight and this pattern of nocturnal maximum and diurnal minimum T _b was most marked between April and September. The circadian T _b cycle was least correlated with photoperiod during winter. Hibernal T _b during winter correlated with ambient temperature (T _a ),it was maximal in September (17.7±1.0°C) and minimal in December (5.2±0.9°C). Apart from the tracking of T _a and T _b during hibernal bouts, with a time-lag of 4–6 h, circadian rhythmicity of hibernal T _b was not evident. However, the T _b of hibernating hedgehogs rose significantly when T _a fell below — 5°C, although the animals did not neccessarily arouse. Although hibernal bouts occurred between September and April, 89.5% of such bouts were recorded between November and February. The mean time of entry into hibernation was 01:45±5.1 h GMT while the mean time of the start of spontaneous arousal from hibernation was 11:53±4.8 h GMT. Therefore, during hibernation hedgehogs were either fully aroused at night, when euthermic hedgehogs have maximalT _b ,or in deep hibernation around midday, when euthermic hedgehogs have minimal T _b .Since wild hedgehogs will feed during spontaneous arousal from hibernation, these timings are probably adaptive, and suggest that entry into, and arousal from, hibernation may be extensions of circadian cyclicity. Spontaneous bouts of transient shallow torpor (TST) were recorded throughout the year, with nearly 80% of observations occurring during August and September, at the start of the hibernal period. TST bouts lasted for 4.9±2.9 h, with T _b falling to 25.8±3.1 °C. Only 20% of TST bouts immediately preceded hibernation and their duration did not correlate with T _a or body mass. TST bouts started at 06:51±4.7 h GMT, significantly later than entry into hibernation, and ended at 13:04±5.4 h GMT. The function of TST bouts is unclear, but they may be preparation for the hibernation season or a further energy conservation strategy. When arousing from hibernation hedgehogs warmed at a rate of 1.9±0.4°C·h^-1, and when entering hibernation cooled at 7.9±1.9°C·h^-1. Warming rates were slightly higher during mid-winter when T _b and body mass were minimal, but cooling rates were 44% higher at the end of the hibernal period compared to the start. Cooling and warming rates were strikingly similar to those measured in hedgehogs at 31°N. These results demonstrate that thermoregulation in the hedgehog is closely regulated and changes on a seasonal basis, in meeting with requirements of surviving food shortages and low temperature during winter.Abbreviations T _a ambient temperature - T _b body temperature - CSD circular standard deviation - SWS slow wave sleep - TST transient shallow torpor     

Thermal acclimation induced compensation in biochemical constituents of the Indian freshwater leech,Poecilobdella viridis (Blanchard)

Poecilobdella viridis was used to study the variations in the biochemical parameters: body protein, glycogen and water, following warm (32.0° ± 0.5°C) and cold (10.0° ± 0.5°C) acclimation for 5, 10, 15 and 30 days. The percentage of glycogen decreased significantly (p < 0.05) during both warm and cold acclimation whereas the protein percentage increased significantly (p < 0.05). The percentage of water was significantly (p < 0.05). reduced during both acclimation processes. Ecophysiological significance of these alterations to leeches, in counteracting the ambient thermal fluxes, has been briefly discussed.